Reversible tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins is a fundamental regulatory process for insulin signal transduction. The long-term goals of this project are to elucidate the signaling roles and cellular regulation of specific protein-tyrosine phosphatases (PTPs) in insulin action and in pathological states of insulin resistance. During the last funding cycle, we have identified a novel regulatory pathway whereby H2O2 generated by cellular insulin stimulation leads to the reversible inhibition of specific PTPs, via oxidation of the catalytic thiol moiety, which enhances receptor substrate tyrosine phosphorylation and facilitates insulin action. In particular, this process involves PTP1B, an intracellular PTP strongly implicated as a negative regulator of insulin signaling; additional oxidation-sensitive signaling proteins are also likely be regulated by this process. We have provided compelling evidence that the NADPH oxidase homolog Nox4 is the source of insulin-stimulated H2O2 and the redox regulation of PTP catalytic activity in insulin-sensitive cells. The current revised proposal will test four major hypotheses: (1) Nox4 plays an integral role in insulin-stimulated H2O2 generation and is coupled to specific molecular components shown to regulate other NADPH oxidases, including p22phox, Rac, Galphai2, NOXO1, NOXA1 or related proteins; (2) Insulin stimulation of NADPH oxidase in isolated plasma membranes is functionally linked to specific Nox regulatory subunits; that high glucose enhances insulin-stimulated cellular H2O2 by PKC stimulation of Nox4; (3) Global loss of Nox4 expression in knock-out mice, or tissue-specific loss of Nox4 in transgenic models leads to impaired insulin signaling due to excess PTP activity in vivo; (4) Insulin-stimulated H2O2 leads to the oxidation and reversible inactivation of a limited set of susceptible cellular proteins involved in insulin signal transduction (including PTPs) that can be identified using novel biochemical reagents. Each of these aims will provide insight into novel regulatory mechanisms that may help identify new targets to enhance insulin sensitivity in common insulin-resistant states such as obesity and type 2 diabetes.